scholarly journals The palmitoylation status of the G-protein Go1 α regulates its activity of interaction with the plasma membrane

1994 ◽  
Vol 302 (3) ◽  
pp. 913-920 ◽  
Author(s):  
M A Grassie ◽  
J F McCallum ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Significant Proportion ◽  
Mutant Form ◽  
Guanine Nucleotide ◽  
Wild Type ◽  
Cytoplasmic Fraction ◽  
Response Curves ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation

Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5′-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.

scholarly journals Inhibition by pertussis toxin of the activation of Na+-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells

1991 ◽  
Vol 280 (2) ◽  
pp. 515-519 ◽  
Author(s):  
J A Sokoloski ◽  
A C Sartorelli ◽  
R E Handschumacher ◽  
C W Lee
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Dimethyl Sulphoxide ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dependent Transport

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).

scholarly journals GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

1988 ◽  
Vol 254 (2) ◽  
pp. 391-396 ◽  
Author(s):  
G Milligan ◽  
I Mullaney ◽  
C G Unson ◽  
L Marshall ◽  
A M Spiegel ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Alpha Subunit ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5′-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of ‘Gi-like’ proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation.

scholarly journals Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells

1988 ◽  
Vol 252 (2) ◽  
pp. 369-373 ◽  
Author(s):  
G Milligan ◽  
F R McKenzie
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Hybrid Cells ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Binding

NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.

scholarly journals Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and α2C10 adrenoceptor-transfected Rat 1 fibroblasts

1994 ◽  
Vol 298 (2) ◽  
pp. 493-497 ◽  
Author(s):  
C Carr ◽  
M Grassie ◽  
G Milligan
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Lysophosphatidic Acid ◽  
Gtpase Activity ◽  
Response Curves ◽  
Protein Activation ◽  
Adp Ribosylation ◽  
High Affinity ◽  
Stimulation Of

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [32P]ADP-ribosylation of Gi. This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [32P]ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein

1992 ◽  
Vol 70 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Richard W. Lerner ◽  
Gary D. Lopaschuk ◽  
Peter M. Olley
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Cyclase Activity ◽  
Prostaglandin E ◽  
Adenylyl Cyclase Activity ◽  
Adp Ribosylation ◽  
Number Of Binding Sites ◽  
Guanine Nucleotide Binding

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 μM 5′-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 ± 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 ± 0.7 nM) to one site of intermediate affinity (KD = 0.52 ± 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein. They also demonstrate that the interaction of this Gi protein with the PGE2 receptor is important in the regulation of PGE2 binding to its receptor.Key words: prostaglandin E2, sarcolemma, heart, adenylyl cyclase, G protein.

Hepatocyte beta-adrenergic responsiveness and guanine nucleotide-binding regulatory proteins

1989 ◽  
Vol 256 (2) ◽  
pp. C384-C389 ◽  
Author(s):  
J. A. Garcia-Sainz ◽  
M. E. Huerta-Bahena ◽  
C. C. Malbon
Keyword(s):  
Binding Protein ◽  
Nucleotide Binding ◽  
Regulatory Proteins ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Beta Adrenergic ◽  
Adp Ribosylation ◽  
Liver Membranes ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

Hepatocytes isolated from hypothyroid, adrenalectomized, or partially hepatectomized rats display an enhanced beta-adrenergic responsiveness as compared with cells from control animals. The enhanced beta-adrenergic responsiveness is evidenced by both increased ureagenesis and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol. The role of stimulatory guanine nucleotide-binding protein (Gs) and inhibitory guanine nucleotide-binding protein (Gi) in the enhanced responsiveness was studied. It was observed, contrary to what would have been anticipated, that the level of Gs [as reflected by cholera toxin-catalyzed ADP ribosylation, 5'-guanosine gamma-thiotriphosphate (GTP gamma S)-stimulated adenylate cyclase activity, and a functional reconstitution assay] was decreased in liver membranes from adrenalectomized and partially hepatectomized rats as compared with the controls. Furthermore, the level of Gi was increased in these conditions as reflected by pertussis toxin-catalyzed ADP ribosylation. The data suggest that changes in beta-adrenergic receptor levels rather than the levels of guanine nucleotide-binding (G) regulatory proteins predominate in regulation of hepatic beta-adrenergic responses by hypothyroidism, adrenalectomy, or partial hepatectomy.

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